Intensity (%) Intensity (%)

(1)

FORUM II

Dispersion und Retention von Ultrafeinstaub/Nanopartikeln in der Lunge

Dr. Otto Creutzenberg Dirk Schaudien, PhD

Fraunhofer-Institut für Toxikologie und Experimentelle Medizin Hannover

Einzelnes Nanopartikel

Agglomerat

Aggregat

Agglomerate aus Aggregat

(2)

Projektausschreibung

Projektausschreibung:

Bundesanstalt für Arbeitsschutz und Arbeitsmedizin (BAuA)

Forschungsprojekt F2133:

Dispersion und Retention von Stäuben mit ultrafeinen Primärpartikeln

in der Lunge

(3)

Gliederung

in vitro Ansätze (TEM)

in vivo Ansätze

• Intratracheale Instillation (TEM, BAL)

• Akute Inhalation (TEM)

TEM: Transmissionselektronenmikroskopie BAL: Bronchoalveoläre Lavage

(4)

TEM analysis

• Fixation in 5% glutaraldehyde solution (pH 7.2)

• PET-membrane was cut out and postfixed in 1% osmiumtetroxid in 0.1M sodium cacodylate buffer.

• Following dehydration of cells in a graded series of ethanol the membranes were infiltrated with epoxy resin and embedded.

• An ultramicrotome (Ultracut E, Richard-Jung) was used for cutting ultrathin sections (70nm).

• Positioning on copper grids and observed with a transmission electron microsope (Zeiss, Leo 910).

(5)

16HBE140- A549

10, 100, 1000 1, 24, 48

(from apical side) Air-liquid

Interface Culture Human

epithelial cell lines Calu-3

Human lung fibroblasts LK004 cells

Particle concentration

(ng/cm²) Exposure

duration (hrs) Method

Cell type Cell line

Selection of Cell Lines

(6)

Rationale for Dosing in vitro

in vivo: 300 µg/lung (no overload conditions)

in vitro: lung surface is approx. 3000 cm²/lung; conversion in vivo Æ in vitro: approx. 100 ng/cm² dosing scheme: 10 - 100 - 1000 ng/cm²

Results

Most useful cell types: A549 and LK004 cells

After 1 hr nanoparticles were predominantly found attached to the cellular surface After 24 hrs these particles were mainly within the cells

(7)

-41 436

0.15 %

Na₂HPO₄buffer 0.1 %

Constantan

n.m.

180 after 24 h

-24 180

0.15 %

Na₂HPO₄buffer 0.1 %

TiO₂T805

n.m.

216 after 17 h

-55 212

0.15 %

Na₂HPO₄buffer 0.1 %

TiO₂P25

ζpotential (mV) Z-Average

(nm) Medium

Concen- tration

(%) Substance /

particle type

Comparison of TiO₂ P25 (hydrophilic) and TiO₂ T805 (hydrophobic)

(8)

Comparison of the diameters of TiO₂P25 particles after 1hr and 24hrs of treatment

Comparison of the diameters of TiO₂T805 particles after 1hr and 24hrs of treatment

Increase of agglomerates No increase of agglomerates

(9)

Gliederung

in vitro

Ansätze (TEM)

in vivo Ansätze

• Intratracheale Instillation (TEM, BAL)

• Akute Inhalation (TEM)

(10)

Diameter of the TiO₂P25 particles at different time points after instillation

Diameter of the TiO₂T805 particles at different time points after instillation

(11)

TiO₂ P25 nanoparticles within a macrophage TiO₂P25 nanoparticle within the

(12)

TiO₂T805 nanoparticles within the lung lining fluid 1 hour after instillation

TiO₂T805 nanoparticles within a macrophage 28 days after instillation

(13)

- Particle suspension medium: Compatible with lung milieu

- Lavage was performed using saline or phosphate buffer (not altering per se the agglomerate status of particles)

- Single lavage only to keep the particle concentration high for ZetaSizer Analysis

TiO₂ P25 TiO₂ T805

Hydrophilic Hydrophobic

Forming rapidly larger agglomerates Remaining at stable agglomerate size

(14)

0 2 4 6 8 10 12

0.1 1 10 100 1000 10000

Intensity (%)

Size (d.nm)

Record 129: T805 Stammlösung 1Tag 1 Record 134: NaCl-BAL nach T805 0,2% 15 min /Att=6 3

0 2 4 6 8 10 12

0.1 1 10 100 1000 10000

Intensity (%)

Size (d.nm) Size Distribution by Intensity

Record 129: T805 Stammlösung 1Tag 1 Record 139: NaCl-BAL nach T805 0,2% 1h /Att=6 2

0 5 10 15 20

0.1 1 10 100 1000 10000

Intensity (%)

Record 129: T805 Stammlösung 1Tag 1 Record 148: NaCl-BAl nach T805 0,2% 4h /Att=6 2

TiO₂T805

15 min

0 20 40 60 80

0.1 1 10 100 1000 10000

Intensity (%)

Record 129: T805 Stammlösung 1Tag 1 Record 153: NaCl-BAL nach T805 0,2% 1d /Att=6 1

1 hr

5 hrs

24 hrs

0 2 4 6 8 10 12

0.1 1 10 100 1000 10000

Intensity (%)

Size (d.nm)

Record 94: P25 0,2% frisch 2 Record 102: PPuffer-BAL nach P25 0,2% 15min Att=6 1

0 5 10 15 20 25

0.1 1 10 100 1000 10000

Intensity (%)

Record 94: P25 0,2% f risch 2 Record 106: PPuf f er-BAL nach P25 0,2% 1h Att=6 2

TiO₂ P25

15 min

1 hr

0 10 20 30

0.1 1 10 100 1000 10000

Intensity (%)

Record 94: P25 0,2% frisch 2 Record 122: PPuffer-BAL nach P25 0,2% 4h Atten =6 3

5 hrs

Precipitation!

(15)

Gliederung

in vitro

Ansätze (TEM)

in vivo Ansätze

• Intratracheale Instillation (TEM, BAL)

• Akute Inhalation (TEM)

(16)

Acute inhalations: Single Exposure of Rats to Various Nanoscaled Aerosols for 6 hrs

I Constantan - spark generator (no ageing) II Constantan - spark generator (ageing)

III Constantan - nebulisation of particle suspension

IV Europiumoxid - nebulisation of particle suspension

(17)

0.E+00 2.E+06 4.E+06 6.E+06 8.E+06 1.E+07 1.E+07 1.E+07

0 100 200 300 400

time [min]

concentration [ 1/cm³ ]

I Acute inhalation Iof a constantan aerosol: Spark generator without ageing

Average number concentration: 9.6·10⁶ [1/cm³]

Average size distribution: 43 nm (mean mobility diamete GSD: 1.9

Assumptions

Minute volume: 0.2 L/min Surface area; 0.4 m² Deposition rate: 50%

Particle loading: 10⁸ [1/cm²]= 1 [1/µm²].

(18)

II Acute inhalation of a constantan aerosol: Spark generator with ageing

Average number concentration: 4.6·10⁵ [1/cm³]

Average size distribution: 124 nm (mean mobility diamet GSD: 1.9

Assumptions

Minute volume: 0.2 L/min Surface area; 0.4 m² Deposition rate: 50%

Particle loading: 6 x 10⁶ [1/cm²]= 0.06 [1/µm²].

0.E+00 1.E+05 2.E+05 3.E+05 4.E+05 5.E+05 6.E+05

0 100 200 300 400

time [min]

concentration [ 1/cm³ ]

(19)

I Acute inhalation of a constantan aerosol (spark generator without ageing)

Constantan particle detected 1 h after end of inhalation in the cytoplasm of an epithelial cell (size: approx. 100 nm)

(20)

Dispersion of constantan particles in a phosphate buffer incl BSA

(0.15 w-% constantan, 0.15 w-% di-sodium-phosphate, 0.15 w-% BSA) Aerosol concentration: 41.6 ± 5.4 mg/m3 (constantan/phosphate/albumin)

13.9 mg/m3 (constantan)

MMAD: 1.37 µm – GSD: 1.53 MPPD model: 6.7% (P)

2.7 % (TB) 50.6% (H)

Deposited mass: approx. 94 µg/6 hrs (P + TB) approx. 67 µg/6 hrs (P)

0 2 4 6 8

0.1 1 10 100 1000 10000

Intensity (%)

Record 236: Kon 0,15%, 0,15%PPuff, 0,15% BSA 40 min Utr St4/ 5. Ansatz 1

0 100000 200000 300000 400000 500000 600000

-200 -100 0 100 200

Total Counts

Zeta Potential (mV) Zeta Potential Distribution

Record 292: Kon 0,15%, 0,15%PPuff , 0,15% BSA 40 min Utr St4/ 5. Ansatz 2

0 5 10 15 20

0.1 1 10 100 1000 10000

Intensity (%)

ζpotential

Impinger (Z-average: 281 nm)

Stock suspension constantan

(21)

2 3 2 3

Dispersion of Eu₂O₃ particles in a phosphate buffer incl BSA

(0.1 w-% Eu₂O₃, 0.15 w-% di-sodium-phosphate, 0.25 w-% BSA) Aerosol concentration: 45.1 ± 7.9 mg/m^{3 (Eu}2O₃/phosphate/albumin)

9 mg/m^{3 (Eu}2O₃)

MMAD: 1.35 µm – GSD: 1.65 MPPD model: 6.1% (P)

2.4 % (TB) 50.7% (H)

Deposited mass: approx. 55.1 µg/6 hrs (P + TB) approx. 39.6 µg/6 hrs (P)

ζpotential

Impinger (Z-average: 281 nm)

Stock suspension constantan

0 2 4 6 8 10

0.1 1 10 100 1000 10000

Intensity (%)

Record 263: Eu2O3 0,1%, 0,15 PPuff, 0,25% BSA 1h Utr St5 /4.Ansatz 1

0 100000 200000 300000 400000 500000 600000 700000

-200 -100 0 100 200

Total Counts

Zeta Potential (mV) Zeta Potential Distribution

Record 289: Eu2O3 0,1%, 0,15 PPuff , 0,25% BSA 65min Utr 2 min Usch / 4. Ansatz 2

10 20 30 40

Intensity (%)

Size Distribution by Intensity

(22)

Urine Blood Heart

0.854 0.272 294

0.088 93.8 32.3

Liver

0.031 0.016 10.8

0.016 5.5 5.7

Kidneys

0.011 0.008 3.9

0.023 2.9 8.3

Spleen

0.009 0.011 3.1

0.006 3.8 2.2

Brain

35,047 34,467

36,779 Lungs

5 days 1 day

1 hour Preserved

organs

% of lungs ng/organ

% of lungs ng/organ

Retained europium oxide per organ - means (n=3)

1 hr after end of exposure: 36.8 µg in lungs = approx. 90% of the amount predicted by the MPPD model.

(23)

Eu₂O₃ nanoparticles within lung lining fluid Eu₂O₃nanoparticles within lung lining

(24)

Zusammenfassung

Agglomeratstatus Suspension

• Herstellung nanoskaliger wäßriger Suspensionen aus den Bulk-Materialien Æ hoher mechanischer/energetischer Aufwand sowie Hilfsstoffe nötig

• Nanopartikel mit hydrophober Oberfläche sind im Lungenmilieu stabiler als gleiche mit hydrophiler Oberfläche (Beispiel: TiO₂ P25 – TiO₂ T805)

Agglomeratstatus Zellen/Lunge

• Nanoskalige Partikel zeigten bei Wechselwirkung mit Zellen (in vitro) oder nach Deposition im Respirationstrakt (in vivo) vorherrschend eine Tendenz zur Bildung größerer

Agglomerate.

• Umgekehrt war ein Agglomeratzerfall nur von geringer Relevanz.

(25)

Danke !

¾

Bundesanstalt für Arbeitsschutz und Arbeitsmedizin (BAuA) Dr. B. Orthen, Prof. T. Gebel

Allen beteiligten Mitarbeiter/innen

am Fraunhofer ITEM

(26)

Projektausschreibung

Bundesanstalt für Arbeitsschutz und Arbeitsmedizin (BAuA)

Forschungsprojekt F2246:

Toxische Wirkungen verschiedener Modifikationen eines Nanopartikel nach Inhalation

Vergleich dreier nanoskaliger Titandioxide im 28-Tage Test

(27)

Vergleich dreier nanoskaliger Titandioxide im 28-Tage Test

600 g Anatas/

Rutil 80/20 TiO₂P25 vom

freien Markt;

charakterisiert von EU

hydrophilic

unbehandelt 60

22 NM-105

600 g Rutil

UV TITAN M262 hydrophilic

Glycerin 60

20 NM-104

600 g Rutil

UV TITAN M212 hydrophobic

Dimethicone (Silikon) 60

20 NM-103

Bestand im ITEM Modifikation

Name Oberfläche

modifiziert mit:

Spez. Oberfläche (m²/g)

PPD (nm) EU/JRC-

Name

TiO₂Proben; charakterisiert und bereitgestellt von der EU Kommission/JRC (Ch. Klein), jeweils Flaschen von 20 ml Volumen, Argon-befüllt, for "in vitro and single use" zu 2 g

(28)

Vergleich dreier nanoskaliger Titandioxide im 28-Tage Test

Design

• Phys.-chem. Charakterisierung, Agglomeratverhalten

• in vitro: Agglomeratverhalten, Zytotoxizität

• in vivo: Pilottest

• in vivo: 28-Tage Inhalationstest

- Zeitpunkte: 1, 45 und 90 Tage nach Expositionsende - Orientierung an OECD 412

- Histopathologie

- Partikelretention in der Lunge - TEM-Analysen